MRC National Institute for Medical Research , The Ridgeway, London NW7 1AA UK.
Sci Rep. 2012;2:919. doi: 10.1038/srep00919. Epub 2012 Dec 4.
Anomalous expansion of a polymorphic tract in Ataxin-1 causes the autosomal dominant spinocerebellar ataxia type 1. In addition to polyglutamine expansion, requirements for development of pathology are phosphorylation of serine 776 in Ataxin-1 and nuclear localization of the protein. The phosphorylation state of serine 776 is also crucial for selection of the Ataxin-1 multiple partners. Here, we have used FRET for an in cell study of the interaction of Ataxin-1 with the spliceosome-associated U2AF65 and the adaptor 14-3-3 proteins. Using wild-type Ataxin-1 and Ser776 mutants to a phosphomimetic aspartate and to alanine, we show that U2AF65 binds Ataxin-1 in a Ser776 phosphorylation independent manner whereas 14-3-3 interacts with phosphorylated wild-type Ataxin-1 but not with the mutants. These results indicate that Ser776 acts as the molecular switch that discriminates between normal and aberrant function and that phosphomimetics is not a generally valid approach whose applicability should be carefully validated.
Ataxin-1 中多态性片段的异常扩展导致常染色体显性小脑共济失调 1 型。除了多谷氨酰胺扩展外，Ataxin-1 丝氨酸 776 的磷酸化和蛋白质的核定位也是发病机制的要求。丝氨酸 776 的磷酸化状态对于 Ataxin-1 多个伴侣的选择也至关重要。在这里，我们使用 FRET 进行了细胞内研究，研究了 Ataxin-1 与剪接体相关的 U2AF65 和衔接蛋白 14-3-3 蛋白的相互作用。使用野生型 Ataxin-1 和 Ser776 突变体（磷酸模拟天冬氨酸和丙氨酸），我们表明 U2AF65 以丝氨酸 776 磷酸化独立的方式与 Ataxin-1 结合，而 14-3-3 与磷酸化的野生型 Ataxin-1 相互作用，但不与突变体相互作用。这些结果表明，Ser776 作为分子开关，区分正常和异常功能，而磷酸模拟物不是一种普遍有效的方法，其适用性应仔细验证。
